Sarah Palmer

Residual viremia can be detected in most HIV-1-infected patients on antiretroviral therapy despite suppression of plasma RNA to R ² = 0.27, P = 0.001 and R ² = 0.19, P < 0.005, respectively), supporting a biological link between the extent of overall baseline viral infection and the infection of long-lived reservoirs. These data suggest that low-level persistent viremia appears to arise from at least two cell compartments, one in which viral production decays over time and a second in which viral production remains stable for at least 7 years.

Single-dose nevirapine (sdNVP) for prevention of mother-to-child transmission of HIV-1 can select nevirapine (NVP)-resistant variants, but the frequency, duration, and clinical significance of this resistance is not well defined. We used a sensitive allele-specific PCR assay to assess the emergence and persistence of NVP-resistant variants in plasma samples from 22 women with HIV-1 subtype C infection who participated in a study of sdNVP for prevention of mother-to-child transmission of HIV-1. The women were categorized into three groups on the basis of detection of NVP resistance by standard genotype analysis. Group 1 ( n = 6) had NVP resistance detected at 2 and 6 mo after sdNVP, but not at 12 mo. Group 2 ( n = 9) had NVP resistance detected at 2 mo, but not 6 mo. Group 3 ( n = 7) had no NVP resistance detected at any time point. Allele-specific PCR analysis for the two most common NVP resistance mutations (K103N and Y181C) detected NVP-resistant variants in most (16 of 21) samples that were negative for NVP resistance by standard genotype, at levels ranging from 0.1% to 20% 1 yr after treatment. The frequency of NVP-resistant mutations decreased over time, but persisted above predose levels for more than 1 yr in ≥23% of the women. These findings highlight the urgent need for studies assessing the impact of sdNVP on the efficacy of subsequent antiretroviral therapy containing NVP or other nonnucleoside reverse transcriptase inhibitors.

Background: Understanding the selection and decay of drug-resistant HIV-1 variants is important for designing optimal antiretroviral therapy. Objective: To develop a high-throughput, real-time reverse transcriptase (RT) polymerase chain reaction (PCR) assay to quantify non-nucleoside reverse transcriptase inhibitor (NNRTI)-resistant variants K103N (AAT or AAC alleles) at frequencies as low as 0.1%, and to apply this to monitor these variants before, during, and after NNRTI therapy. Methods: HIV-1 RNA in longitudinal plasma samples obtained from patients starting and stopping NNRTI therapy was converted to cDNA and the target sequence region amplified and quantified by real-time PCR. Approximately 10⁷ copies/reaction provided a template for a second round of PCR using primers that discriminated between the mutant and wild-type alleles. Amplification specificity was confirmed by thermal denaturation analysis. Results: Frequencies of 103N similar to assay background (0.029%) were observed in longitudinal samples from 9 of 12 treatment-naive patients; three patients had transient increases in 103N frequency to a range of 0.21–0.48%, which was 7–16.5 times assay background. Analysis of longitudinal plasma samples from six NNRTI-experienced patients showed three patterns: persistence of 103N variants after stopping NNRTI therapy, codon switching of 103N between AAC and AAT during NNRTI therapy, and decay of 103N variants to below assay background after cessation of NNRTI therapy. Conclusions: Allele-specific RT-PCR quantified the emergence and decay of drug-resistant variants in patients over a broad range of frequencies (0.1–100%). The rate of decay of K103N variants after stopping NNRTI therapy was highly variable.

To investigate the extent to which drug resistance mutations are missed by standard genotyping methods, we analyzed the same plasma samples from 26 patients with suspected multidrug-resistant human immunodeficiency virus type 1 by using a newly developed single-genome sequencing technique and compared it to standard genotype analysis. Plasma samples were obtained from patients with prior exposure to at least two antiretroviral drug classes and who were on a failing antiretroviral regimen. Standard genotypes were obtained by reverse transcriptase (RT)-PCR and sequencing of the bulk PCR product. For single-genome sequencing, cDNA derived from plasma RNA was serially diluted to 1 copy per reaction, and a region encompassing p6, protease, and a portion of RT was amplified and sequenced. Sequences from 15 to 46 single viral genomes were obtained from each plasma sample. Drug resistance mutations identified by single-genome sequencing were not detected by standard genotype analysis in 24 of the 26 patients studied. Mutations present in less than 10% of single genomes were almost never detected in standard genotypes (1 of 86). Similarly, mutations present in 10 to 35% of single genomes were detected only 25% of the time in standard genotypes. For example, in one patient, 10 mutations identified by single-genome sequencing and conferring resistance to protease inhibitors (PIs), nucleoside analog reverse transcriptase inhibitors, and nonnucleoside reverse transcriptase inhibitors (NNRTIs) were not detected by standard genotyping methods. Each of these mutations was present in 5 to 20% of the 20 genomes analyzed; 15% of the genomes in this sample contained linked PI mutations, none of which were present in the standard genotype. In another patient sample, 33% of genomes contained five linked NNRTI resistance mutations, none of which were detected by standard genotype analysis. These findings illustrate the inadequacy of the standard genotype for detecting low-frequency drug resistance mutations. In addition to having greater sensitivity, single-genome sequencing identifies linked mutations that confer high-level drug resistance. Such linkage cannot be detected by standard genotype analysis.

More sensitive assays for human immunodeficiency virus type 1 (HIV-1) RNA are needed to detect, quantify, and characterize persistent viremia in patients who are receiving antiretroviral therapy and whose plasma HIV-1 RNA levels are suppressed to less than 50 to 75 copies/ml. We therefore developed an internally controlled real-time reverse transcriptase-initiated PCR assay that quantifies HIV-1 RNA concentrations down to 1 copy per ml of plasma. This assay with single-copy sensitivity (the single-copy assay) generates a reproducible linear regression plot of input copy number versus threshold cycle by using HIV-1 RNA transcripts at copy numbers ranging from 1 to 10 ⁶ per reaction mixture. The single-copy assay was compared to the ultrasensitive AMPLICOR HIV-1 MONITOR assay and a more sensitive modification of the ultrasensitive assay by repeatedly testing a low-copy-number panel containing 200 to 0.781 copies of HIV-1 RNA per ml of plasma. This comparison showed that the single-copy assay had a greater sensitivity than the other assays and was the only assay that detected HIV-1 RNA at levels as low as 0.781 copies/ml. Testing of plasma samples from 15 patients who were receiving antiretroviral therapy and who had <75 HIV-1 RNA copies/ml revealed persistent viremia in all 15 patients, with HIV-1 RNA levels ranging from 1 to 32 copies/ml (median, 13 copies/ml). The greater sensitivity of the single-copy assay should allow better characterization of persistent viremia in patients who are receiving antiretroviral therapy and whose HIV-1 RNA levels are suppressed to below the detection limits of present assays.

We analyzed the reverse transcriptase (RT) and protease sequences of HIV-1 isolates obtained over 7 years from two couples with known transmission histories. Phylogenetic trees constructed from the sequence data reflected the known transmission histories, despite the fact that the drug resistance mutations were most consistent with the drug treatment histories. However, the RT sequences from one couple diverged by 2.9% even before therapy was begun, and three (0.9%) of 339 unrelated individuals had viruses that shared a common ancestor with sequences from the recipient member of the couple but not with sequences from the transmitter. The divergence between the first two isolates from this couple is consistent with a pretransmission interval during which the transmitter developed a heterogeneous virus population. The closeness between the three controls and the recipient's first RT sequence may indicate slower evolution on the branches of the control sequences. Although the RT and protease genes contain phylogenetic information, they are suboptimal for reconstructing transmission history because the genetic distance between RT and protease isolates from unrelated individuals may occasionally approximate the distance between RT and protease isolates from related individuals. Address correspondence and reprint requests to Sarah Palmer, PhD, HIV Drug Resistance Program, National Cancer Institute at Frederick, 1050 Boyles Street, Building 535, Room 109, Frederick, MD 21702-1201, U.S.A.; e-mail: [email protected] Manuscript received May 22, 2002; accepted July 23, 2002. © 2002 Lippincott Williams & Wilkins, Inc.

New antiretroviral drugs with activity against strains of human immunodeficiency virus type 1 (HIV-1) with non-B subtypes and with resistance to current antiretroviral drugs are needed. The activity of two nucleotide analogs, tenofovir and adefovir (PMPA and PMEA, respectively), against non-B subtypes and nucleoside-resistant primary HIV-1 isolates was assessed. Tenofovir and adefovir were fully active against a panel of subtypes A, C, D, E, F, G, and group O primary HIV-1 isolates as compared with their respective activity against subtype B isolates. Moreover, the susceptibility of a panel of 10 primary HIV-1 isolates with >10-fold mean resistance to zidovudine, lamivudine, and abacavir was within 2.2-fold of wild-type tenofovir susceptibility for each isolate. An oral prodrug of tenofovir, tenofovir disoproxil fumarate (DF), is currently in phase III clinical trials for the treatment of HIV-1 infection. These in vitro susceptibility results suggest that tenofovir DF may be active in vivo against HIV-1 with nucleoside resistance as well as against HIV-1 with non-B subtypes.